Preservation of dried blood grouping serum



Aug. 29, 19. A. F. cocA 2,357,253

I PRESERVATION OF DRIED BLOOD GROUPING SERUM Filed Aug. 1, 1940 7 2 INVENTOR.

BY w w fQ Q\ 1 ATTORNEY.

Patented Aug. 29, 1944 SATS PRESERVATION F DRIED BLOOD GROUPIN G SERUM tion of Delaware Application August 1, 1940, Serial 'No. 349,053 Claims. (Cl. 252-40 8) The present invention relates to blood grouping sera and is more particularly directed to a method and container for preserving desiccated blood grouping sera.

In the transfusion of blood it is first necessary to determine the blood group of boththe patient and the prospective blood donor in order that their blood may match. Such determinations are ordinarily made using sera of persons of group 2 and group 3 which are respectively specific reagents for the two corpuscular group substances B and A. These sera areusually supplied in liquid form in hermetically sealed capillary tubes and if stored in an ice chest remain potent. It has not been found practical to distribute the sera in containers holding more than a very small quantity, as even the addition of preservatives will not prevent the sera from losing their potency. Because of the difiiculties thus encountered in preserving and distributing the liquid blood grouping sera, the cost is not only exorbitant, but it is also diflicult to have readily available quantities of the testing serum at all times. I It has been found that high-titer group-specific sera can be obtained by immunizing lower animals for example with group AB corpuscles. The rabbit is especially suited for this work. The resulting rabbit serum, after its absorption with group B corpuscles, is effective against group A corpuscles. The production of serum effective against group B corpuscles can likewise be obtained by treating the rabbit serum with group A corpuscles.

As an alternative method in determining blood groups, desiccated sera may be employed instead of the sera in liquid form. These desiccated sera may be produced from either fluid serum obtained from human or the fluid serum obtained by the immunization of the lower animals such as rabbits. Various methods have been devised for desiccating the serum without destroying the agglutinating factors. One of the most widely employed prccess'es involves freezing the serum and subjecting to a vacuum while in the frozen state to produce a desiccated serum. The present invention, however, is not primarily concerned with the manner in which the blood grouping serum is desiccated and any method which produces a dried serum without affecting its special biologic activity may be used, but it is particularly concerned with a method of rendering the dried blood grouping serum stable under practical conditions.

In using the desiccated serum in blood grouping tests, the desiccated powder is usually diluted with distilled water to restore it to about its original consistency and the test carried outvin the same manner as when employing serum in liquid form. The desiccated serum possesses many advantages over the fluid serum, it being more easily distributed and handled, and theamount of serum needed for a test is exceedingly small. While the desiccated serum is mor desirable to use under conditions which are not entirely satisfactory for using the liquid serum, there are, however, some serious disadvantages possessed by the dried serum. The dried serum is very hygroscopic and when exposed to the atmosphere for a time it becomes moist and rapidly loses its potency. In the past these dried sera have been preserved by storing in an evacuated container and at a low temperature under which conditions they can be satisfactorily preserved for some time. If the containers are not kept tightly closed, the serum cannot be preserved at ordinary temperatures, and once the container has been opened the serum has a strong tendency to absorb moisture and its agglutinating power is destroyed.

In accordance with the present invention I have discovered that desiccated blood: grouping sera can be diluted with dried powdered substances, suchas sucrose, dextrose, sodium chloride, or other substantially non-hygroscopic water-soluble salts which do not affect the agglutinating power of the sera, and that this diluted serum is stable if stored in a dry atmosphere. The diluted serum is much less hygroscopic than the undiluted serum, and hence is not subject to as rapid deterioration in the presence of appreciable quantities of moisture. The

diluted serum canbe stored in an ordinary con-' tainer at room temperature provided the container is not permitted to remain open or exposed to a moist atmosphere for a considerable period of time, and it is not necessary that it be stored in an evacuated container at low temperatures.

- I have found, however, that when the diluted serum is stored in an atmosphere which is constantly kept dry, the container can be opened from time to time and portions of the serum re moved for making blood grouping tests and yet the remaining serum remains stable over a long period of time and maintains its agglutinating potency. In a preferred embodiment of the present invention the diluted serum is stored in a compartment of a container which is constantly kept dry by some drying agent, such as dried calcium chloride.

The method of diluting the desiccated serum the diluent used and upon the source and quality of the desiccated serum. In most instances I have found that good results are obtained whenv one part of the dried serum is diluted with about parts of the diluent. On the other hand, satisfactory results may be obtained when the dilution ratio is about 1:1 or 1:50. If desired, the diluted desiccated sera may be colored with different dyes in order to maintain a clear line of distinction between groups 2 and 3. Any of the many suitable dyes available may be used. I have found, for example, that eosin can be added to the anti-B which gives a' red color and that methylene blue can be added to the anti-A to give a blue color. Only small amounts of the dye need be used. c

A container suitable for storing the diluted dried serum is illustrated by the drawing in which: 9 Fig. 1 is a perspective view of the container;

and

Fig. 2 'is alongitudina section through the container showing the relative position of the diluted serum andthe dessicating substance. 1

Referring more specifically to the drawing, 1 represents a vial or a container consisting of a glass tube about 2 /2 inches long and 12 mm. in diameter. A tightly fitting cotton dam 2 is held in about the center portion 'of the tube and is flanked on either side by a layer of porous or tissue paper 3, thus dividing the tube into two compartments 4 and 6. The compartment 4 isgfilled with a suitable drying agent, such as calcium chloride, and the stopper 5 is inserted in the opening, preferably at such a distance that it is flush with the end of the tube. The other compartment 8 contains the desiccated diluted serum. The end of this compartment is closed by means of the stopper 1' which fits tightly but is inserted in such a manner that it is convenient for withdrawing and replacing.

It is apparent from examination of the drawing that the diluted desiccated serum contained in the compartment 6 is protected against moisture and that the atmosphere surrounding the "dried material is constantly being subjected to the drying action of the calcium chloride contained in the compartment 4. The layer of paper! prevents the desiccated serum from coming in direct contact with the cotton plug 2 and prevents the calcium chloride from sifting through the cotton plug to contaminate the diluted serum. The cotton plug 2 may of course be replaced with any other suitable material e. g. glass wool or asbestos fibers which are porous to vapors. Cotton is preferred, however, because it is a relatively poor absorbent for moisture and hence will permit moist vapors to pass readily into the calcium chloride.

In one specific instance the compartment 6 was filled to about one half its capacity with a mixture comprising 1 part of a dried blood grouping serum and 20 parts of dry powdered cane sugar and the compartment 4 was filled with anhydrous calcium chloride, and the container was then stored at room temperature. The potency of the dried serum was tested from time to time. At the end of about a year and a half the serum had suffered no perceptible loss of potency. Similar tests were conducted for shorter periods of time using different dilutions of serum and sucrose and in which the containers were stored at temperatures varying from that found in an ice-box up to about 62 C., and in all cases the serum retained its potency.

In place of the anhydrous calcium chloride other drying agents may be used, such as anhydrous calcium sulfate, anhydrous aluminum I chloride, or any of the ordinary dehydrating salts.

There are a great many advantages resulting from the useof the composition and packaging arrangement of the present invention. For example, it is well known that macroscopic grouping tests of blood depend on the use of serums having extremely high hemagglutinin titer and that a number of fluid serums are not suitable for use because oftheir lowtiter value. In order to use these liquid serums of low titer they are usually mixed-withaserum having a high titer toproduce one of medium titer but suitable for use. In thecaseof a desiccated serum, it is possible to convert a liquid serum of. relatively low titer to one of satisfactory high titer. For example, if 200 cc. of the fiuid serum were desiccated and this desiccated serum diluted to only 50 cc. when carrying out the actual test, the agglutinating power of the desiccated serum would be approximately four times that of the fluid serum.

It is also an advantage that the diluted desiccated blood grouping sera of this invention need not be stored in an evacuated container and at a low temperature. They can be stored in a non-evacuated chamber in an atmosphere dried by a drying agent and at room temperature. It is a further advantagethat the combination of the diluted dried serum and the container provide a package whichwill permit portions of the serum to be removed in making tests and the diluted serum left in the container will remain useful for carrying out such tests over a long period of time.

What I claim is: v I

1. A stable composition comprising a dry mixture of about one part by weight of desiccated blood serum containing one of the active blood agglutinants of the group consisting of anti-A and anti-B and one to fifty parts by weight of a dry, water-soluble, inert diluent which does not; affect the .agglutinating action of the agglutinant, said composition being soluble in water to provide a blood grouping solution of high titer.

.2. A stable composition comprising a dry mixture of about one part by weight of desiccated blood serum containing one of the active blood agglutinants of the group consisting of anti-A and anti-B and one to fifty parts by weight of dry sucrose, said composition being soluble in water to provide a blood grouping solution of high titer.

I 3. A stable composition comprising a dry mixture ofabout one part by weight of desiccated blood serum containing one of the activeblood agglutinants of the group consisting of anti-A and anti-B and oneto fifty parts by weight of dry dextrose, said composition being soluble in water to provide a blood grouping solution of high titer.

,4. A stable composition comprising -a dry mixture of about one part by weight of desiccated blood serum containing one of the active blood agglutinants of the group consisting of anti-A and anti-B and one to fifty parts by weight of dry sodium chloride, said composition being soluble in water to provide a blood grouping solution of high titer.

5. A stable composition comprising a dry mixture of about one part by weight of desiccated blood serum of lower animals containing one of the active blood agglutinants oi the group consisting of anti-A and anti-B, one to fifty parts by weight of a dry. water-soluble, inert diluent which does not affect the agglutinating action of the agglutinant, and a small amount of a dye, said composition being soluble in water to provide a blood grouping solution of high titer.

ARTHUR. F. COCA. 

